This invention relates to a novel protein and, more particularly, to a highly purified mammalian transcription factor given the name E2F-2.
The mammalian transcription factor E2F was originally identified as a cellular factor recruited by adenovirus Type 5 to initiate transcription from the viral E2 promoter (for a review see J. R. Nevins, Science, 258:424-429 1992)). It has subsequently been suggested that E2F may play a critical role in cell-cycle regulation of uninfected mammalian cells, as well. E2F binding sites are found in the promoter regions of several cellular genes which are important for cell growth, including c-myc, cdc2, and DHFR. E2F activity appears to be regulated via complex formation with other cellular proteins in a cell-cycle dependent fashion. Binding of E2F to the retinoblastoma gene product (pRb) yields a complex which suppresses transcription of genes containing the E2F binding site. Only the underphosphorylated form of pRb is found in the E2F/pRb complex, and the complex is present in the G.sub.1 stage of the cell cycle and persists into the S phase. Interactions of E2F with other cellular proteins, including the pRb homolog p107 and cyclins, suggest that a complicated set of growth regulatory functions are mediated by these proteins.
Clones of two distinct proteins with E2F-like activity have recently been identified. Human E2F-1 was identified by probing expression libraries with recombinant pRb (W. J. Kaelin et al., Cell, 70:351-364 (1992); B. Shan et al., Mol. Cell. Biol., 12:5620-5631 (1992); K. Helin et al., Cell, 70:337-350 (1992)). This protein displays many of the properties of authentic E2F, including binding to the E2F recognition element in a sequence-specific fashion and binding to pRb. Furthermore it has been shown that the DNA and pRb binding activities of purified HeLa cell E2F and recombinant E2F-1 are enhanced by heterodimerization (H. E. Huber et al., Proc. Natl. Acad. Sci. USA, 90:3525-3529 (1993)). Following preparative SDS-PAGE of affinity-purified E2F, two sets of proteins were identified, and one component from each set was required for optimal reconstitution of activity. It has also been demonstrated that recombinant E2F-1 forms homodimers, and proposed that these homodimers may be responsible for the observed DNA and pRb binding activity of E2F-1 in the absence of its normal partner. A distinct protein (DP-1) was more recently cloned based on amino acid sequence data obtained from E2F purified from mouse F9 cells (R. Girling et al., Nature, 363:83-87 (1993)). This cloned protein also displays the key biochemical properties of cellular E2F, but displays little sequence homology to E2F-1 outside of the DNA binding domain.
Isolation, purification, identification and expression of proteins having E2F-like activity is useful in studying the mechanisms of cell proliferation and for screening for compounds which would inhibit such activity and, consequently, inhibit abnormal cell proliferation.